Seed storage protein with nutritionally balanced amino acid composition

ABSTRACT

The present invention relates, in general to protein that is a seed storage protein having high nutritional value. In particular, the invention relates to the protein AmA1 and to a DNA sequence encoding same. The invention further relates to a recombinant molecule comprising the AmA1 encoding sequence and to a host cell transformed therewith. In addition, the invention relates to a method for producing transgenic plants with high nutritionally rich amino acids.

This is a division of application Ser. No. 08/158,270 filed Nov. 29,1993 now U.S. Pat. No. 5,670,635.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates, in general, to a seed storage proteinfrom Amaranthus hypochondriacus with nutritionally balanced amino acidcomposition. In particular, the invention relates to the AmA1 (AmaranthAlbumin 1) protein and to a DNA sequence encoding same. The inventionfurther relates to a recombinant molecule comprising AmA1 encodingsequence and to a host cell transformed therewith. In addition, theinvention relates to a method for improving the nutritional quality ofsome crop plants.

2. Background Information

Seed storage proteins, intended as a source of nitrogen for germinatingseedlings, form an important source of dietary protein for human beings.A balanced composition of amino acids is therefore required in the humandiet, but most often seeds are deficient in one or the other essentialamino acids. For years plant breeders have tried to improve the balanceof essential amino acids of the important crop plants (Larkins, B. A.(1993) in Genetic Engineering of Plants: An Agricultural Perspective(Plenium, N.Y.), pp. 93-118). Molecular approaches for improving thenutritional quality of seed proteins provide alternatives to theconventional approaches. Attempts in vitro mutagenesis of the codingregion of certain seed proteins has been tried to increase the levels ofessential amino acids (Larkins, supra and Hoffman, L. M., Donaldson, D.D. & Herman, E. M. (1988) Plant Mol. Biol. 11, 717-729). Anotherapproach is to transfer heterologous storage protein genes that encodeproteins with higher levels of limiting amino acids (Guerche, P., DeAlmeida, E., Schwarztein, M. A., Gander, E., Krebbers, E. & Pelletier,G. (1990) Mol. Gen. Genet. 221, 306-314). Expressing high levels of aparticular amino acid by heterologous gene transfer or by mutation maybe detrimental to the normal physiology of seed development. This mayalso produce seeds with a biased amino acid composition. An alternativeapproach will therefore be to express a gene for a heterologous proteinwith a balanced amino acid composition.

Grain Amaranth is a pseudo cereal with high protein content (17-19% ofseed dry weight) as compared to more traditional crops which have anaverage of about 10% protein (Davies et al (1980) in The Biochemistry ofPlants (Academic Press, New York)). Its protein is rich in essentialamino acids like lysine, tryptophan and sulphur amino acids (Senft, J.P. (1980) in Proceedings of the 2nd Amaranth conference (Rodale Press,Pennsylvania), pp. 43-47), that are otherwise deficient in the majorseed proteins of legumes and cereals. It can therefore be used as asource of a gene that encodes a protein of high nutritional value. Inspite of good quality and quantity, these proteins have not yet beenpurified and characterized. In Amaranthus, 50% of the total seedproteins at maturity are globulin and albumin (Paredes-Lopez, O.,Mora-Escobedo, R. & Ordorica-Falonir, C. (1988) Lebensm. Wiss U-Technol.21, 59-61). The present invention provides purified AmA1 (Albumin seedstorage protein) and a DNA sequence encoding same. The invention alsoprovides a step toward developing transgenic plants with a balancedamino acid composition.

OBJECTS OF THE INVENTION

An object of the invention is to propose a protein that is a seedstorage protein having nutritional value.

Another object of this invention is to isolate, characterize andconstruct a gene that can be used in the expression of albumin seedstorage protein in plants.

A further object of this invention is to propose a recombinant moleculecomprising the AmA1 encoding sequence and to a host cell transformedtherewith.

It is a further object of this invention to introduce AmA1 gene intoplants such as field crops such as rice, maize, wheat and legumes,vegetables crops such as carrot, potato, etc., thereby improving thenutritional quality of such plants.

BRIEF DESCRIPTION OF INVENTION

The present invention is broadly directed to the use of a seed storageprotein, as exemplified by AmA1, for commercial uses such as in thebrewing industry or for agronomic uses such as to improve human dietaryprotein by introducing the gene into major cereal and legumes.

The present invention also relates to AmA1 substantially purified andcharacterized. The AmA1 is an albumin protein with a pH value of 6.8.The protein is a 35 kDa, monomer with four isoforms that can beseparated by chromatofocussing. AmA1 is a cytosolic protein. Unlike anyother seed storage proteins neither is it localized in protein bodiesnor does it have any glycosylation.

The present invention also relates to substantially all of asubstantially pure gene encoding a seed storage protein AmA1 with aspecific DNA sequence as shown in SEQ ID NO:1:. The gene encodes analbumin protein exhibiting high nutritional quality having a molecularweight of 35 kDa, as determined by SDS-PAGE.

The invention relates to compositions for use in raising transgenicplants, which compositions have balanced amino acid, in particular AmA1protein expression. Specifically, the protein has a high proportion ofessential amino acids such as lysine, leucine, threonine, phenylalanine,valine and sulfur amino acids that are otherwise deficient in major seedproteins of legumes and cereals. The AmA1 amino acid composition iscomparable to the WORLD HEALTH ORGANIZATION (WHO) recommended values fora highly nutritional protein, making it more important nutritionally. Anagronomic use for such a protein is to combine the compounds with anappropriate carrier, which is agronomically acceptable, permittingdelivery of the compound directly to the plant or to the soil in whichthe plants grow.

A transformed plant cell is also disclosed herein, which cell istransformed with a gene encoding AmA1 protein. The gene encoding such aprotein can include the DNA sequence set forth in SEQ ID NO:1:.

Further objects and advantages of the present invention will be clearfrom the description of the invention that follows:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D Purification of 35 kDa protein. (FIG. 1A) Elution profile ofthe Chromatofocussing column; , A₂₈₀ ; ₋₋₋₋₋₋, pH; I-IV, protein peaks.The unnumbered peak before peak I did not contain protein. (FIG. 1B)SDS/PAGE analysis column fractions. L, material loaded; W, wash; M,molecular mass standards, Numbers indicate the fractions analyzed. Arrowindicates the 35 kDa protein. Equal volumes of each fraction wereanalyzed. (FIG. 1C) Purity of AmA1 by one-dimensional gelelectrophoresis. The purified protein 10 μg (lane 1) and 5 μg (lane 2)of the purified protein was analyzed on 12% SDS/PAGE and stained withCoomassie Blue. Lane 3 shows molecular mass standards. (FIG. 1D) Purityof AmA1 by two-dimensional gel electrophoresis. Protein (5 μg) wasanalyzed in the first dimension by SDS/PAGE on a 10% gel and stainedwith Coomassie Blue. Dark patch at the bottom of the gel contains theampholytes.

FIG. 2 Western blot analysis of peak fractions from a chromatofocussingcolumn probed with AmA1 antibodies. Equal volumes of sample fromfractions obtained after chromatofocussing were resolved by SDS/PAGE ona 12% gel and transferred to nitrocellulose. Immunoreactive polypeptideswere detected with AmA1 antibodies. Lanes; A, Albumin fraction and I-V,peak fractions (FIG. 1A) analyzed by SDS/PAGE; C, albumin fractionanalyzed on an isoelectric focussing gel prior to immunodetection.Arrows indicate the various isoelectric forms. Fourth band is very faintand thus not very clear.

FIGS. 3A and 3B Analysis of subclones. (FIG. 3A) Inserts from lambdaclones 2, 3, and 5, were subcloned in pTZ18U vector. All three cloneswere digested with EcoRI and analysed on a 2% agarose gel. Clone numbersare shown above the lanes. (FIG. 3B) Hybrid-selected translation productof subclone 3 was immunoprecipitated with AmA1 antibodies bound toprotein A-Sepharose and analyzed by SDS/PAGE on a 12% gel. The ³⁵ Slabeled and immunoprecipitated 35 kDa protein was detected byautoradiography.

FIGS. 4A-4D Expression of AmA1 gene during seed development. (FIGS. 4Aand 4B) Crude extract (20 μg of Protein) of seeds at various stages ofdevelopment, as in Table I, were resolved by SDS/PAGE on a 12% gel induplicate. Protein bands in one gel were stained with Coomassie Blue(FIG. 4A) and in the other were subjected to Western blot analysis (FIG.4B). (FIGS. 4C and 4D) Total RNA was extracted from developing seeds and2-ug sample were separated on a 1.2% agarose gel containing glyoxal induplicate. RNA integrity was checked by ethidium bromide staining (FIG.4C). AmA1-specific mRNA was identified by Northern blot analysis. RNAwas transferred to GeneScreenPlus and probed with labeled AmA1 cDNA(FIG. 4D).

FIGS. 5A ad 5B Seed-specific expression of AmA1 gene. Total RNA wasisolated from the following tissues. (FIG. 5A) Lanes: 1, seedling; 2,seed; 3, root; 4, leaf. (FIG. 5B) Lanes: 1, leaf; 2, root; 3, seed; 4,1-day-old seedling. (FIG. 5A) Ethidium bromide-stained gel to show theintegrity of rRNA bands. Arrows indicate the rRNA bands. (FIG. 5B)Northern blot probed with labeled AmA1 cDNA.

FIG. 6--Sequencing strategy and the restriction map of AmA1 cDNA. Thedirection of transcription and length of the open reading frame isindicated by the solid thick arrow. The short thin horizontal arrowsindicate the sequencing strategy used in both orientations.

FIG. 7--Hydropathic plot of the AmA1 protein. The 304-amino acidsequence of the predicted AmA1 protein was analyzed by the method ofKyte and Doolittle with a window setting of 7.

FIG. 8--Position of intron in genomic clone of AmA1. The forward primerand the reverse primer were positioned and shown by arrow heads. Thejunction of the intron start and end sites were shown by the sequencesmentioned.

FIGS. 9A and 9B--Nucleotide sequence of the 1183-bp AmA1 cDNA and thederived amino acid sequence of the predicted polypeptide. Amino acids(single-letter code) are indicated above the first base of each codon.Possible glycolation sites are overlined. It does not show poly (A)tract although poly (A) addition signals are indicated and areunderlined.

The purification of AmA1 from Amaranthus hypochondriacus is described inthe Examples below, as is the characterization of the isolated protein.The four forms of the protein were resolved on chromatofocussing columnat pH 7.4, 7.1, 6.8 and 6.7. Protein eluting at pH 7.4, AmA1 (peak 1)was further purified on a gel filtration column. FIGS. 1A, 1C and 1Dshow the purity of the protein by one and two dimensional gelelectrophoresis. Antibodies raised against purified peak 1 proteinshowed immunoreactivity with the 35 kDa polypeptide present in otherpeaks, indicating that the protein may have at least four isoforms (FIG.2). Analysis of the albumin fraction on an isoelectric focussing gelvisible on immunostaining (FIG. 2, Lane C) further confirms theexistence of four isoforms. The protein is a 35 kDa water-soluble,non-glycosylated monomer with four isoforms, having an acidic pH.Analysis of RNA and protein in developing seeds showed that AmA1 issynthesized during early embryogenesis (FIGS. 4A and 4B), reaching amaximum by midmaturation. No RNA was detected in one day old seedlingsalthough the protein showed delayed breakdown on germination, thusindicating its developmental regulation. Mature seeds even after oneyear of storage contained AmA1 mRNA, although at reduced levelssuggesting that it is very stable.

The gene encoding the plant derived AmA1 protein having the sequenceshown in SEQ ID NO:1: was cloned as described in the Examples thatfollow. Briefly, the cDNA encoding the protein was obtained byimmunoscreening a lambda gt11 expression library. In vitro translationof hybrid-selected mRNA gave a 35 kDa protein. Genomic Southernhybridization indicated that AmA1 is encoded by a single gene. Affinitypurified AmA1 antibodies were used to isolate cDNA clones fromdeveloping-seed expression library. The six immunopositive plaquesrecombinant obtained were found to be related. The cDNA of the largestclone (1.2 kb) has a single major open reading frame corresponding to a304-amino acid polypeptide. The clone was confirmed by hybrid-selectedtranslation and immunoprecipitation.

The gene having the structure of SEQ ID NO:1: containing the codingsequence for the mature AmA1 protein can be attached to geneticregulatory elements that are needed for the expression of the structuralgene in a defined host cell. The first type of regulatory elementrequired is a gene promoter region, which contains DNA sequencesrecognized by the biological machinery of the plant cell and whichinduces transcription of the DNA sequence into messenger RNA (mRNA). ThemRNA is then translated into the protein product coded for by thestructural gene region. The promoter is attached in front of or 5' tothe gene for AmA1, which can be performed according to standard methodsknown in the art. See, for example, Maniatis et al, (1982) MolecularCloning, Cold Spring Harbor Laboratory, New York, pp. 104-106.

Promoter regions which can be used for expression of the AmA1 gene inplant cells include promoters which are active in a wide range ofdifferent plant tissues. For example, the 35S promoter from thecauliflower mosaic virus may be suitable for this purpose. Another typeof promoter that can be used in plant cells is one that expresses undermore restricted conditions. Included in this class are promoters activeonly in certain tissue(s) of the plant and/or induced to be active bycertain stimuli like wounding. An example of this kind of promoter isthe 5' regulatory region from the gene for gluteline, patatin, RUBPcase.These types of promoters are discussed in (Takaiwa et al., Plant Mol.Biol. 16, 49-58, 1991). Expression of the AmA1 gene in yeast hosts canbe achieved by use of promoters obtained from yeast sources. Examples ofsuch promoters include the gal, adh promoter for expression in yeastsuch as YEP51, pAAH5 as exemplified in Gustav Ammerer, In methods inEnzymology, Vol. 101, pp. 192-201 & Broach et al., ExperimentalManipulation of gene expression, pp.83-117. The gene promoter sequencescan also be derived in part or in whole from promoter sequences found incells unlike those of the host cells as long as they meet the abovecriteria for transcription and translation.

A second genetic regulatory element which desirably can be, but need notbe, attached to the AmA1protein gene is a terminator or polyadenylationsequence that promotes effective termination of transcription of thegene and, in eukaryotes, also promotes polyadenylation, i.e., theaddition of any number of adenosine nucleotides at the 3' end of themRNA. Standard methods known in the art can be used to attach theterminator region behind or 3' to the gene. (See, for example, T.Maniatis et al, supra, pp. 104-106). An example of such aterminator/polyadenylation sequence for expression in plants is thatfrom the octopine synthase gene and or nopaline synthase gene from anAgrobacterium tumefaciens Ti plasmid as enunciated in DeGreve et al,(1982), J. Mol. Appl. Genet. 1: 499-511. The gene terminator sequencescan also be derived in part or in whole from terminator sequences foundin cells unlike those of the host cell, as long as they meet the abovecriteria for transcription termination and polyadenylation required bythe host cell.

Another type of regulatory element which can be attached to the gene forAmA1 is a DNA sequence coding for a signal peptide. The signal peptideis attached the amino terminus of the protein and permits the protein tobe localized inside the protein bodies or secreted from the host cell.During this localization process, the signal peptide is cleaved off,producing a protein product with the sequence of the mature protein. TheDNA sequence for the signal peptide is inserted between the promoter andthe coding region. Standard methods known in the art may be used toattach the DNA sequence for the signal peptide (see, for example,Maniatis, T., et al, supra, pp. 104-106). Examples of such signalsequences include the signal peptide from a patatin gene and orgluteline gene of plants (Rosahl et al., Mol. Gen Genet. 203, 214-220,1986) and from prepro factor of yeast (Smith et al, 1985, Science 229:1219-1229). The signal peptide sequences can also be derived in whole orin part from signal sequences found in cells unlike those of the hostcell, as long as they meet the above criteria for processing andlocalization of the protein in the host cell.

Any of the various methods known for introducing foreign genes intoplants can be used for insertion of AmA1 gene into a host plant. Themethodology chosen to accomplish plant transformation with the AmA1 genevaries depending on the host plant. By way of example, onewell-characterized methodology that would be useful for planttransformation with AmA1 gene is Agrobacterium mediated transformation.

Agrobacterium mediated transformation using the AmA1 gene follows theprocedure well-known for this methodology. First, a gene cassettesuitable for expression in plants is introduced into a disarmed strainof Agrobacterium tumefaciens as in intermediate host. The AmA1 genecassette is introduced into the T-DNA region of a recombinant plasmidcontaining a selectable marker gene such as a gene encoding for neomycinphosphotransferase II, phosphinothricin acetyl transferase, or the like.This methodology is set forth in many literature publications includingHorsch et al, (1985), Science 227: 1229-1231. Pieces of plant tissue,e.g. leaf, cotyledons or hypocotyl are co-incubated with the bacteriafor 2-3 days before the bacteria are killed using antibiotics such ascarbenicillin. Additional antibiotics corresponding to the selectablemarker gene employed are included in the plant tissue culture mediumsuch that only transformed plant cells will grow.

Plants regenerated from the transformed cells are then tested for thepresence and expression of the AmA1 gene. Immunoassays and tests forAmA1 protein activity can be used to identify individual transformants.

As noted, several other methodologies are available for planttransformation apart from Agrobacterium transformation. Examples ofthese other DNA delivery methods include electrophoration, i.e.chemically induced delivery into protoplasts, micro-injection,biolistics, as well as others. Examples of types of plants that are notespecially suitable for Agrobacterium-mediated transformation arelegumes and certain cereals including rice. These plants would plainlybenefit from plant transformation attempts using biolistic approaches.

Certain aspects of the present invention will be described in greaterdetail in the non-limiting Examples that follow.

EXAMPLE 1 PURIFICATION AND CHARACTERIZATION OF AMA1 PROTEIN

Experimental Protocols:

Plant Material

Seeds of Amaranthus hypochondriacus were obtained from National Bureauof Plant Genetic Research, Simla, India. Mature seeds were ground to afine powder and defatted by extraction with cold acetone. Seeds and thedefatted seed meal were stored at 4° C. under desiccation. Seeds atdifferent developmental stages were kept frozen until use.

Purification and Characterization of 35 kDa albumin

Defatted seed meal (1 g) was extracted with 10 ml of buffer A (25 mMTris acetate, pH 8.5) containing 1M NaCl and 2 mM Phenyl methyl sulfonylfluoride. Homogenate was centrifuged and the supernatant was dialyzedagainst buffer A. Precipitate formed on dialysis was removed bycentrifugation and the supernatant was chromatographed on aDEAE-Sepharose column (1×25 cm; 25 ml/h), pre-equilibrated with bufferA. Column was washed with the same buffer and the bound protein waseluted with a multicomponent buffer system, as suggested by Prestidgeand Hearn (Anal. Biochem. 97, 95-102, 1979) with slight modifications.Fractions (2 ml) were collected and analyzed on SDS/PAGE. Appropriatefractions, after analysis on SDS/PAGE, were concentrated using centricon(Amicon) and subjected to further purification on a Sephadex G-75(1.6×30 cm) column previously equilibrated with buffer A containing 0.1MNaCl. Elution was carried out in the same buffer at a flow rate of 10ml/h. Fractions were analyzed on SDS/PAGE and pure protein wasconcentrated and stored at 4° C.

SDS/PAGE and NEPHGE

Purity of the sample was routinely checked by SDS/PAGE using 12%separating and 4% stacking gel according to the procedure of Laemmli(Nature 227, 680-685, 1970). The gels were stained with CoomassieBrilliant Blue R-250. Non-equilibrium pH gradient electrophoresis(NEPHGE) was performed by the procedure of O'Farrell et al. (J. Biol.Chem. 250, 4007-4021, 1975). Purified protein or crude albumin fractionwas separated by NEPHGE in the first dimension using Pharmalyte pH 3-10(Pharmacia). The gel was equilibrated in SDS sample buffer prior tosecond dimension gel.

Antisera preparation and immunodetection

Antibodies against purified AmA1 were raised in rabbits. They wereaffinity purified essentially by the procedure of Elledge and Davis(Mol. Cell. Biol. 7, 2783-2793, 1987). Western blot analysis was done asdescribed by Towbin et al. (Proc. Natl. Acad. Sci. USA 76, 4350-4354,1979). Protein bands were visualized by staining with Ponceau S(Salinovich, O. & Montelaro, R. (1986) Anal. Biochem. 156, 341-347)prior to immunodetection. Antibodies were used at a dilution of 1:10,000(crude antibodies) or 1:5,000 (affinity-purified antibodies). Alkalinephosphatase conjugated anti-rabbit IgG (Promega) was used as secondaryantibody.

Amino acid analysis The amino acid content of the purified protein wasdetermined by the LKB Alpha plus 4151 amino acid analyser. Samples werehydrolysed with 6N HCl under vacuum at 105° C. for 22 h prior toanalysis.

RNA isolation

Total RNA was isolated by Phenol/Chloroform extraction and LiClprecipitation procedure described by Ausubel et al. (Current Protocolsin Molecular Biology, Greene Publishing Associates andWiley-Interscience, New York, pp. 4.3.1-4.3.3, 1987). Poly(A)⁺ RNA wasisolated from total RNA by two rounds of selection on oligo (dT)cellulose according to the procedure of Okayama et al. (In MethodsEnzymol. 154, 3-28, 1987).

in vitro translation and immunoprecipitation

RNA (2 μg) or Poly(A)⁺ RNA (0.5 μg) was translated in a rabbitreticulocyte lysate system (Promega) and immunoprecipitated according tothe procedure of Anderson and Blobel (Methods Enzymol. 96, 111-120,1983) except that antibodies were bound to protein A-Sepharose beads in0.1M sodium phosphate buffer, pH 8.0 prior to immunoprecipitation.Immunoprecipitated product was boiled in SDS sample buffer andelectrophoresed. Gel was stained and fluorography was done (Chamberlain,J. P. (1979) Anal. Biochem. 98, 132-135). It was exposed to X-Ray filmat -70° C. Hybrid selected translation of AmA1 mRNA was done accordingto the procedure of Ricciardi et al (Proc. Natl. Acad. Sci. USA 76,4927-4931, 1979).

Results:

Albumin fraction obtained on dialysis of the crude extract waschromatographed on chromatofocussing column. On chromatofocussing, 35kDa albumin protein eluted in four different peaks at pH values of 7.4,7.1, 6.8 and 6.7 (a faint band but clearly visible on immunoblotting(FIGS. 1A & 1B); other two peaks did not have any 35-kDa albuminprotein. Protein eluting at pH 7.4, AmA1 (peak I), was maximum and wasfurther purified on a gel filtration column (data not shown). FIGS. 1Cand 1D show the purity of the protein by one and two dimensional gelelectrophoresis. Antibodies raised against purified peak I proteinshowed immunoreactivity with the 35 kDa polypeptide present in otherpears, indicating that the protein may have at least four isoforms (FIG.2). When albumin fraction was analysed on IEF gel, three distinct bandsand a fourth faint band were visible on immunostaining (lane C, FIG. 2).It further confirmed the existence of isoforms.

EXAMPLE 2 MOLECULAR CLONING AND EXPRESSION OF DNA ENCODING AMA1 PROTEIN

Experimental Protocols:

cDNA construction and screening

Poly(A⁺) RNA isolated at the stages when AnA1 mRNA was high (stages IIIand IV, Table I) was used as a template for cDNA synthesis. cDNA wassynthesized by the method of Gubler and Hoffman (Gene 25, 263-269, 1983)and cloned in lambda gt11 according to Young and Davis (Proc. Natl.Acad. Sci. USA 80, 1194-1198, 1983). Briefly, first strand cDNAsynthesis was carried out using reverse transcriptase and oligo (dT) asprimer (Amersham cDNA synthesis kit). cDNA was sequentially treated withS1 nuclease, EcoRI methylase and Klenow fragment of DNA polymerase Iprior to blunt end ligation to EcoRI linkers. Linkered cDNA was ligatedto EcoRI ended lambda gt11 arms (Amersham cDNA cloning kit). ResultingDNA was packaged in vitro and used to infect E. coli strain Y1090. About17000 recombinant phages were plated on two 150 mm plates, induced withIPTG and fusion proteins were detected by immunoscreening using AmA1antibodies.

Subcloning of insert DNA

Lambda DNA was purified by the CTAB procedure of Manfioletti andSchneider (Nucleic Acids Res. 16, 2873-2884, 1988). Insert was purifiedfrom the EcoRI digested recombinant lambda DNA and ligated to EcoRI cutpTZ18U and M13mp18 vector DNA. E. coli strains JM101 and JM109 served ashosts for plasmid and phage vectors respectively. Plasmid and M13 DNAwas purified by standard protocols (Sambrook, J., Fritsch, E. F. &Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, New York).

cDNA sequence analysis

Sequencing was done in M13mp18 DNA in both orientations by the Dideoxychain termination method using Sequenase Version 2.0 (United StatesBiochemical Corporation). To read the complete cDNA sequence, deletionswere generated in both orientations using Exonuclease III and S1nuclease (Sambrook, J., Fritsch, E. F. & Maniatis, T., Supra.Orientation of the insert in the lambda gt 11 clone was directlydetermined in lambda gt11 by using lambda gt11 primers in sequencing.

Northern blot analysis

Total cellular RNA (10 μg) was denatured with glyoxal and separated byelectrophoresis on 2% agarose gel containing glyoxal. The amount of RNAand the integrity of rRNA was confirmed by ethidium bromide staining ofa duplicate gel. Gel was blotted onto Gene Screen plus membrane (DuPont)and probed with AmA1 cDNA labeled to a specific activity of about 3×10⁸cpm/μg DNA (Feinberg, A. & Vogelstein, B. (1984) Anal. Biochem. 137,266-267).

PCR Aided Genomic cloning

Genomic DNA was isolated from seeds by the procedure of Rogers & Bendich(In Plant Mol. Biol. Manual, eds: Gelvin S B & Schilperoort R A, KluwerAcademic Publishers).

The genomic DNA was amplified to get the genomic clone of AmA1 bypolymerase chain reaction using one forward primer and another reverseprimer.

The forward primer was designed and made such that after amplificationthe amplified fragment will have the starting ATG. PCR was done in a 25μl reaction volume using 350 ng of genomic DNA as template with Taq DNApolymerase by Perkin Elmer Cetus and components from GeneAmp® kit. Theamplification was done in a PTC-100 - 60 thermal cycler of M.J.Res.Inc., (USA). The reaction mixture was made according to manufacturerinstruction. This PCR amplified Genomic fragment was then cloned inpBluescript ks⁺ at EcoRV site by the procedure of Marchuk D. (NucleicAcid Research, 5: 1154, 1991).

Results

Molecular Cloning of AmA1 cDNA:

cDNA clones corresponding to AmA1 were isolated from a cDNA libraryconstructed in an expression vector lambda gt11. Level of AmA1 mRNA indeveloping seeds was analysed by in vitro translation andimmunoprecipitation. Poly (A)⁺ RNA, from the stages when the level ofAmA1 mRNA was high, was used as the template for cDNA synthesis. A totalof 35,000 plaques were obtained of which six were immunopositive.Positive plaques were selected and subjected to successive rounds ofphage titering and screening to get clonally pure recombinant plaques.DNA was isolated and Southern hybridization was done to see therelatedness of all the clones. Three of the clones designated AmA1.2,AmA1.3 and AmA1.5 were subcloned in plasmid vector pTZ18U. pAmA1.2 andpAmA1.3 had large inserts of 1.2 kb each and pAmA1.5 had an insert of0.25 kb (FIG. 3A). pAmA1.3 was used for hybrid selected translation.Translated product when immunoprecipitated and analysed on SDS/PAGE gavea polypeptide of 35 kDa that comigrated with the 35 kDa band of purifiedAmA1 (FIG. 3B). This band was absent when no exogenous RNA was presentin the translation reaction or when vector alone was used forhybridization.

Developmental regulation and seed specific expression of AmA1:

Expression of most seed protein genes is regulated in time and in space.To study the developmentally regulated expression of AmA1 gene, totalprotein and RNA from seeds at different developmental stages wereanalysed. Seeds of Amaranthus appear in glomerules. At a particular timepoint in each glomerule seeds at various developmental stages areencountered. Seeds were therefore grouped into developmental stages bytheir weight (Table I). AmA1, as analysed by SDS/PAGE and Western blotanalysis was seen to be synthesized very early on embryogenesis (FIGS.4A & 4B). Total RNA when subjected to Northern blot analysis also showedthe presence of AmA1 mRNA during early embryogenesis (FIGS. 4C & 4D).Mature seeds showed low levels of AmA1 RNA and no RNA was detected inone day old seedlings. Protein level was seen to increase in proportionto the RNA level until stage IV (Table I, FIGS. 4A-4D) after which therewas no further increase in protein in spite of the presence of AmA1mRNA. AmA1 was not detected in other plant parts (data not shown).Northern blot analysis did not show any trace of AmA1 mRNA either inleaves or in roots (FIG. 5). Therefore, these results suggest that theexpression of AmA1 is seed specific.

                  TABLE I                                                         ______________________________________                                        Average weight of seeds at different stages of development                    STAGE        SEED WEIGHT (in mg)                                              ______________________________________                                        I            0.1                                                              II           0.2                                                              III          0.3                                                              IV           0.4                                                              V (Mature)   0.8                                                              ______________________________________                                    

Sequence analysis of AmA1 cDNA

The largest insert of 1.2 kb (from pAmA1.3) was subcloned in M13mp18 inboth orientations to get single stranded DNA. The sequencing strategy issummarized in FIG. 6. The sequenced cDNA (FIG. 7A) has a length of 1183base pairs with an open reading frame (ORF) of 912 bp with non-coding 5'and 3' flanking sequences. The ORF encodes a protein of 304 amino acidswith Mr of 35000 and pH of 6.8. Analysis of the amino acid sequenceshows that it is a hydrophilic protein with a small stretch ofhydrophobic amino acids at the N-terminus (FIG. 7B). Amino acidcomposition as predicted from the cDNA sequence shows high levels ofessential amino acids. It is similar to the composition obtained byprotein hydrolysis (Table II). The differences observed are due totechnical limitations with Picotag system of amino acid analysis as itnormally shows reduced levels of sulphur amino acids and high levels ofglycine. Poly A stretch is missing from the sequence though two putativepoly-adenylation signals are located at 50 and 194 bp downstream of thestop codon (FIG. 7A). No homology was seen between the deduced proteinsequence of AmA1 cDNA and other seed specific proteins.

                  TABLE II                                                        ______________________________________                                        Amino acid composition of AmA1                                                Residues, mol %                                                               Amino          Purified                                                                              cDNA                                                   acid           Protein sequence                                               ______________________________________                                        Phe            5.6     5.6                                                    Tyr            4.8     4.9                                                    Leu            7.6     8.6                                                    Ile            5.0     6.6                                                    Val            5.6     6.2                                                    Met            1.6     2.3                                                    Cys            0.7     1.6                                                    Ala            5.3     4.6                                                    His            3.3     2.3                                                    Thr            5.3     5.9                                                    Pro            3.6     3.6                                                    Gly            12.0    5.3                                                    Glx            8.9     9.2                                                    Asx            16.6    14.5                                                   Ser            6.6     6.9                                                    Arg            5.3     2.6                                                    Lys            6.6     6.9                                                    Trp            *       2.3                                                    ______________________________________                                         *not determined                                                          

The amino acid composition as obtained from purified protein and alsowhat is predicted from the cDNA sequence shows a high proportion ofessential amino acids like lys, leu, thr, phe, val and sulfur aminoacids (Table III) that are otherwise deficient in the major seedproteins of legumes and cereals. It has a relatively low level ofglutamine as compared to other seed storage proteins (Higgins, T. J. V.(1984) Annu. Rev. Plant Physiol. 35, 191-221). Interestingly, AmA1composition closely matches the values recommended by WHO making it moreimportant nutritionally.

                  TABLE III                                                       ______________________________________                                        Percentage of essential amino acids of AmA1 in                                comparison to the World Health Organization recommended values.               % of total amino acids                                                               Amaranth                                                                      Proceedings of                                                                            AmA1          WHO                                                 the Second  Calculated by consi-                                                                        Proceedings of                                      Amaranth Confer-                                                                          dering total residue                                                                        the Second Amar-                             Amino  ence  Senft, J.P                                                                          number of each amino                                                                        anth Conference                              acids  (1980). Rodale,                                                                           acid from the sequence                                                                       Senft, J.P(1980)                            Total  Emmaus, PA; and their respective                                                                        Rodale, Emmaus,                              Protein                                                                              pp. 43-47)  molecular weights.                                                                          PA; pp. 43-47)                               ______________________________________                                        Trp    1.4         3.6           1.0                                          Met/   4.4         3.9           3.5                                          Cys                                                                           Thr    2.9         5.1           4.0                                          Ile    3.0         6.1           4.0                                          Val    3.6         5.2           5.0                                          Lys    5.0         7.5           5.5                                          Phe/   6.4         13.7          6.0                                          Tyr                                                                           Leu    4.7         9.2           7.0                                          ______________________________________                                         WHO, World Health organization.                                          

Molecular Cloning and Sequence analysis of PCR amplified genomic clone

The PCR amplified genomic clones were picked up by colony hybridizationusing Duralon UV membrane according to manufacturer instruction withAmA1 cDNA as probe. The positive clones were having 2.5 kb insertcomprises of the open reading frame interrupted by an intronapproximately 1.5 kb in length and some part of poly(A) tail (FIG. 8).The intron is being sequenced by the dideoxy chain termination methodusing Vent DNA polymerase and components from CircumVent™ Thermal CycleSequencing Kit (New England Biolabs, USA). To read the completesequence, deletions were generated using exonuclease III and S1 nuclease(Sambrook, J., Fritsch, E. F. & Maniatis, T., supra.

Seed storage proteins are localized within protein bodies and are oftenglycosylated. AmA1 was found to be present in the cytosol and notlocalized within protein bodies. This was confirmed by cDNA analysis.The putative polypeptide encoded by the ORF of AmA1 cDNA is 35 kDa. Inaddition, AmA1 mRNA on hybrid selected translation gave a polypeptide of35 kDa that matched the size of the purified protein (FIG. 3B).Hydropathy plot of the deduced protein sequence indicates the presenceof a small stretch of hydrophobic amino acids near the N-terminus (FIG.7). Any possible functional relevance for this region is not yetestablished. This does not appear to be a signal peptide as the aminoacids following this stretch do not meet the requirement of a signalsequence (Perlman, D. & Halvorsen, H. O. (1977) J. Mol. Biol. 115,675-694 and Von Heijne, G. (1984) J. Mol. Biol. 173, 243-251). Threeputative glycosylation sites are also present in the sequence though theprotein did not show any glycosylation by PAS staining (data not shown).AmA1 gene is expressed during early embryogenesis. Mature seeds evenafter one year of storage showed the presence of AmA1 mRNA, though thelevel was reduced. It suggests that it is very stable. Germinated seedsdid not show any AmA1 mRNA. No RNA was detected in leaves and roots,suggesting that the expression is tissue specific. Such specificity ofexpression is due to sequences in the promoter and enhancer regions ofthe gene that we are now looking into.

To summarize, it can be said that AmA1 is a seed specific protein,different from the traditional seed storage proteins. Because of itshigh nutritional value gene encoding this protein may have potential forcompensating for amino acid deficiencies of many seed proteins once itis genetically engineered into target plants.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1183 base pairs                                                   (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATCAGATTAACATAATTTCACAATAAAAAAAAAAAAAAGAGCTTAA46                              ATGGCGGGATTACCAGTGATTATGTGCCTAAAATCAAATAAC88                                  MetAlaGlyLeuProValIleMetCysLeuLysSerAsnAsn                                    1510                                                                          AACCAGAAGTACTTAAGATATCAAAGTGATAATATTCAACAA130                                 AsnGlnLysTyrLeuArgTyrGlnSerAspAsnIleGlnGln                                    152025                                                                        TATGGTCTTCTTCAATTTTCAGCTGATAAGATTTTAGATCCA172                                 TyrGlyLeuLeuGlnPheSerAlaAspLysIleLeuAspPro                                    303540                                                                        TTAGCTCAATTTGAAGTCGAACCTTCCAAGACTTATGATGGT214                                 LeuAlaGlnPheGluValGluProSerLysThrTyrAspGly                                    455055                                                                        CTTGTTCACATCAAATCTCGCTACACTAACAAATATTTGGTT256                                 LeuValHisIleLysSerArgTyrThrAsnLysTyrLeuVal                                    606570                                                                        AGGTGGTCTCCCAATCATTATTGGATTACAGCATCAGCCAAT298                                 ArgTrpSerProAsnHisTyrTrpIleThrAlaSerAlaAsn                                    7580                                                                          GAACCAGATGAAAATAAAAGCAATTGGGCATGCACATTATTC340                                 GluProAspGluAsnLysSerAsnTrpAlaCysThrLeuPhe                                    859095                                                                        AAACCACTTTACGTAGAAGAAGGTAACATGAAAAAGGTTCGA382                                 LysProLeuTyrValGluGluGlyAsnMetLysLysValArg                                    100105110                                                                     CTTTTGCACGTCCAATTAGGTCATTATACAGAAAATTATACC424                                 LeuLeuHisValGlnLeuGlyHisTyrThrGluAsnTyrThr                                    115120125                                                                     GTTGGTGGGTCCTTCGTATCATACTTATTTGCCGAATCAAGT466                                 ValGlyGlySerPheValSerTyrLeuPheAlaGluSerSer                                    130135140                                                                     CAAATTGATACCGGCTCTAAAGACGTATTCCATGTCATAGAT508                                 GlnIleAspThrGlySerLysAspValPheHisValIleAsp                                    145150                                                                        TGGAAATCAATCTTTCAATTTCCCAAAACATATGTCACATTT550                                 TrpLysSerIlePheGlnPheProLysThrTyrValThrPhe                                    155160165                                                                     AAAGGAAATAATGGAAAATATTTAGGGGTTATCACAATTAAT592                                 LysGlyAsnAsnGlyLysTyrLeuGlyValIleThrIleAsn                                    170175180                                                                     CAACTTCCATGTCTACAATTTGGGTATGATAATCTTAATGAT634                                 GlnLeuProCysLeuGlnPheGlyTyrAspAsnLeuAsnAsp                                    185190195                                                                     CCAAAGGTGGCTCATCAAATGTTTGTCACTTCTAATGGTACT676                                 ProLysValAlaHisGlnMetPheValThrSerAsnGlyThr                                    200205210                                                                     ATTTGCATTAAATCCAATTATATGAACAAGTTTTGGAGACTC718                                 IleCysIleLysSerAsnTyrMetAsnLysPheTrpArgLeu                                    215220                                                                        TCTACGGATAATTGGATATTAGTCGATGGGAATGATCCTCGC760                                 SerThrAspAsnTrpIleLeuValAspGlyAsnAspProArg                                    225230235                                                                     GAAACTAATGAAGCTGCTGCGTTGTTTAGGTCGGATGTGCAT802                                 GluThrAsnGluAlaAlaAlaLeuPheArgSerAspValHis                                    240245250                                                                     GATTTTAATGTGATTTCGCTTTTGAACATGCAAAAAACTTGG844                                 AspPheAsnValIleSerLeuLeuAsnMetGlnLysThrTrp                                    255260265                                                                     TTTATTAAGAGATTTACGAGTGGTAAGCCTGAGTTTATAAAT886                                 PheIleLysArgPheThrSerGlyLysProGluPheIleAsn                                    270275280                                                                     TGTATGAATGCAGCTACTCAAATTGTTGATGAAACTGCTATT928                                 CysMetAsnAlaAlaThrGlnIleValAspGluThrAlaIle                                    285290                                                                        TTAGAGATAATAGAATTGGGATCCAACAACTAATATATTGGATT972                               LeuGluIleIleGluLeuGlySerAsnAsn                                                295300                                                                        GCTTTTAAGATTCAAATTAAAGTCTAGTTGTTAATGTAAGGAATAAAACG11022                       TTGTAAGTCGTCTCTTTGGAAACAAGAGGGTTCTTCCTTGTATCATATCT11072                       CTATGGTCTCTTTCAGATTTTGACCATAAGATTACTATTAAATACTTGTA11122                       ATGTGTTTGTCTGTGATGATTACTCTTTGTTGGAATAAAATAATTGTTAG11172                       AATTATATTAC11183                                                              __________________________________________________________________________

We claim:
 1. A process for the preparation of DNA encoding the seedspecific AmA1 protein from Amaranthus which comprises:i) isolatingmessenger RNA from Amaranthus by conventional methods; ii) preparingfrom mRNA double stranded cDNA with reverse transcriptase using oligo(dT) primers; iii) combining the double stranded cDNA with an expressionvector; iv) isolating from the resulting recombinant vectors thoseencoding said seed specific protein by conventional methods; and v)sequencing the DNA encoding said seed specific protein.
 2. A process asclaimed in claim 1 wherein the isolation of messenger RNA from total RNAof Amaranthus seeds is done by oligo (dT) cellulose chromatography.
 3. Aprocess as claimed in claim 1 wherein the synthesis of the doublestranded cDNA is effected by reverse transcriptase using poly(A)⁺ RNA astemplate and oligo (dT) 12-18 as primer to obtain full length cDNA.
 4. Aprocess as claimed in claim 1 wherein the expression vector used is adisarmed bacterial virus.
 5. A process as claimed in claim 1 wherein thedouble stranded cDNA is grown as phage plaques and recombinant proteinsexpressed by phages are transferred to filters and the recombinant cloneidentified using antibodies raised against said seed specific Ama1protein.